protein whose N-terminal methionine is stable and not removed by methionine aminopeptidase. Changing the. Al:U72 base pair to a Gl:C72 base pair greatly re-.
Vol. 268, No. 33, Issue of November 25, pp. 25221-25228,1993 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
The Roleof Nucleotides Conserved in Eukaryotic Initiator Methionine tRNAs in Initiation of Protein Synthesis* (Received for publication, June 1, 1993)
Harold J. Drabkin, Bernhard HelkS, and Uttam L. RajBhandary Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Mutant human initiator tRNA genes carrying changes in eachof the three featuresunique to eukaryotic initiator tRNAs have been constructed, and introduced into CV-1 monkey kidney cells using SV40 virus vectors. The mutant tRNA genes are expressed, and the mutant tRNAs can all be aminoacylated with both rabbit liver and Escherichia colimethionyl-tRNA synthetases. Based on aminoacylation levels, the tRNAs are expressed to 5-15-fold over the level of endogenous initiator tRNA. The activity of the mutant [a6S]methionyl-tRNAsin initiation wasstudied in rabbit reticulocyte and wheat germ cell-free protein synthesis systemsprogrammed with various mRNAs. Initiation is studied by using a mRNA that codes for a protein whose N-terminal methionine is stable and not removed by methionine aminopeptidase. Changing the Al:U72 base pair to a Gl:C72 base pair greatly reduced activity of the tRNA in initiation. Changing the three consecutive G:C base pairs (G29G30G31: C39C4OC41) in the anticodon stem to those found in elongator methionine tRNA also reduced initiation activity. Interestingly, changing the A54 and A60 residues in loop IV to T54 and U60 had less of an effect on activity. The tRNA with changes in all three conserved features had virtually no activity ininitiation.
Protein synthesis in all organisms is initiated with a methionine codon. Cellshave two different species of methionine accepting transfer RNAs, the initiator and the elongator. The initiator methionine tRNA is used in theinitiation of protein synthesis, whereas the elongator methionine tRNA is used for insertion of methionine intointernal peptidic linkage (Kozak, 1983). Because of their unique function,initiator tRNAs have several highly specific properties which are distinct from those of elongator tRNAs. In eukaryotes, these special properties of the initiator methionyl-tRNA include: (i) the formation of a highly specific ternary complex with the initiation factor eIF-2 and GTP, (ii) direct binding to the ribosomal “P” site, and (iii), exclusion from binding to the ribosomal A site. In contrast, theelongator methionyl-tRNA forms a ternary complex with the elongation factor EF-1 and GTP and binds to theribosomal “A” site (reviewedin Moldave (1985), Kozak (1992), and Merrick (1992)).We are interested *This workwas supported by American Cancer Society Grant NP114 and National Institutes of Health Grant GM46942. Parts of this work have been reported before in preliminary form, at the XI1 International Workshop on tRNA, Umea, Sweden (1987). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ Fellow of Deutscher Akademischer Austauschdienst during this study. Present address: Sandoz Pharmaceuticals, Switzerland.
in identifying the sequence and/or structural features in the initiator tRNA that account for these distinctive properties. Along with these special properties, initiatortRNAs of eukaryotes also have three unique featuresnot found in elongator methionine tRNAs. These include: 1) an Al:U72 base pair at the end of the amino acid acceptor stem (RajBhandary and Ghosh, 1969), 2) three consecutive G:C pairs in the anticodon stem, and 3)A54 and A60 in loop IV instead of T54 andpyrimidine 60 conserved in almost all other tRNAs (Simsek and RajBhandary, 1972; Simsek et at., 1973; Sprinzl et al., 1991). To investigate the relationships betwe2n these conserved features and the unique properties of the initiator tRNAs, we have used mutagenesis of a human initiator tRNA gene (Drabkin and RajBhandary, 1985~).We have constructed mutants in which each of these conserved features have been changed, either alone, or in combination, to sequences found in the mammalian elongator methionine tRNA. The mutanttRNAs are obtained by expressing the tRNA genes in CV-1 cells during a lytic infection of SV40 recombinant viruses carrying these genes (DrabkinandRajBhandary, 1985b). Weshow that mutant tRNA genes with up to eight changes are expressed and that they yield tRNA that is still aminoacylated with methionine. In uitro functional studies show that mutation of the Al:U72 base pair to Gl:C72 has a strongeffect on initiation. Mutation of the highly conserved G:C base pairs in the anticodon stem also effects initiation, the effect being more pronounced in the rabbit reticulocyte cell-free system than in the wheat germ system. Surprisingly, mutation of A54 and A60 to T54 and T60 results in only a modest reduction in activity in initiation.’ EXPERIMENTALPROCEDURES
Enzymes and Reagents Crude Escherichia coli aminoacyl-tRNA synthetasewas a gift of T. Y. Hai, and purified E. coli methionyl-tRNA synthetase was a gift of P. Rosevear (University of Houston). Crude rabbit liver aminoacyltRNA synthetase was prepared essentially as described by Pearson et al. (1973). Micrococcal nuclease-treated reticulocyte lysates were obtained from Promega. Wheat germ extracts were provided by M. Sachs. Purified rabbit liver initiator andelongator methionine tRNAs were kind gifts of G . Petrissant. DNAs The M13mp7 clone carrying the wild type human initiator tRNA on a 140-base pair BamHI fragment and the T54 mutant have been described previously (Drabkin and RajBhandary, 1985~).Recombinant SV40 vectors pSVIGT3 andpSVrINS7 are also as described by Drabkin and RajBhandary (198513). The oligodeoxyribonucleotides used for mutagenesis were synthesized on an Applied Biosystems Throughout this paper, the mutant tRNAs and their genes are referred to using the mutations made a t the DNAlevel.Insome cases, the nucleotides are further modified at the RNAlevel;for example U54 to T54, and u31 andU39 to q.
25221
Human Methionine Initiator
25222 Human tRNA
yet
tRNA Human tRNA,”’
:OH
8OH
C
C A
A
-U
GI -pA
6°C
C72
+
G - C C - G A - U G-C A-U G-C
u
C GA
G
T60 I
I I I I
G
A A G C G u
m2G C GA cm7G U -A A29 G C -T41 G-C T31-G - C -T39 C A C t C A U
-
-
-
4
C U A C C A A m’A 1 1 1 1 1 G
m’G C GCm2G
C - G C-G U “G C-G m2G C U-A
-
U
C A C U C
‘”
I I I l l
A D G A C G C m 2 GA G I II I G D A GCGCm2,
G U G A G T m 5 ~
m‘A G C
9
D
T54
$ . - A A G mG ’ C-G A-U G-C
-
*-*
C
U
A t6A
cmA
FIG. 1. Sequence of the human initiator and elongator methionine tRNAs. Arrows indicate the nucleotides changed at the DNA level.
1
2
3
4
5
6
7
8
9
DNA synthesizer and purified by polyacrylamide gel electrophoresis and Sep-pak chromatography (Lo etal., 1984). Generation of Mutants
FIG.2. Expression of mutant tRNA genes. CV-1 cells infected with viruses carrying the various human initiator tRNA genes were labeled with [RzP]orthophosphate as described under “Experimental Procedures,” and the tRNA was analyzed on a 15% polyacrylamide (ratio of acrylamide to bisacrylamide 19:1), 7 M urea gel. Lane 1, RNA from mock infected cultures; lane 2, wild type initiator tRNA; lane3, T54T60; lane 4, Gl:C72; lane 5, Gl:C72/T4T60; lane 6, A29T31:T39T41; lane 7, Gl:C72/A29T31:T39T41; lane 8, A29T31:T39T41/T54T60; lane 9, Gl:C72/A29T31:T39T41/T54T60.
Oligonucleotide-directed Site-specific Mutagenesis-This was performed usingeither theprocedure outlined inZoller and Smith(1982) or the gapped duplex method of Kramer et al. (1984). The mutagenic primers ranged in size from 15to 33 nucleotides. Mutants were identified by hybridization screening using the mutagenic oligonucleotide as a probe (Wallace et al., 1979; Wood et al., 1985), and were verified by sequencing (Sanger etal., 1977). Recombinant SV40 Virus Stocks-The mutant tRNA genes were subcloned into the plasmid pSVlGT3, a late region-defective SV40 hybrid vector (Drabkin and RajBhandary,1985b). In some cases, the genes were cloned into an SV40 vector which does not require helper functions for successful infection.’ Generation of virus stocks following transfection of CV-1 cells with the recombinant SV40 vectors carrying the various mutant tRNA genes was essentiallyas described previously (Drabkin and RajBhandary, 1985b). Analysis of Expression in CV-1 Cells-In vivo labeling with [”PI orthophosphate and analysis and purification of the labeled RNAs by polyacrylamide gel electrophoresishave been detailed previously (Drabkin and RajBhandary,1985b), exceptthat in some labelings the phosphate-free Dulbecco’s modified Eagle’s medium (Sigma) was supplemented with 25 mM Hepes, pH 7.2, to aid in pH control during incubation a t atmospheric CO? levels. Analysis of TI- and T2-RNase digests of R’P-labeled tRNAs was as described elsewhere (Drabkin and RajBhandary, 1985b). Isolation of Unlabeled Total tRNA-Four to five 15-cm dishes of CV-1 cells were infected for 48 h with 2 ml of secondary virus stocks of the various mutants. Transfer RNA was isolated from these cultures asdescribed before (Drabkin, 1988). The average yield from one 15-cm dish of CV-1 cells was about 100 pg of total tRNA. The tRNA was then separated on 15%, 7 M urea polyacrylamide gels (1.5 X 200 X 400 mm), and the overexpressed tRNA was visualized by UVshadowing. The desired tRNA-containing bandwas then excised from the gel and eluted overnight in 0.1 M sodium acetate, pH 6, 10 mM EDTA, 1% phenol(Leontiset al. 1988). The gel fragments were removed by centrifugation through Centrexfilters, and the RNA was then recovered by ethanol precipitation. tRNA preparationsused for incubation in wheat germ extracts were obtained from native polyacrylamide gels. In thiscase, the RNAswere visualized using “Stainsall” (l-ethyl-2[3-(l-ethyInaphtho[l,2d]thiazolin-2-ylidene)-2-methylpropenzyl]naphtho(l,2d]thiazoliumbromide.
* H. J. Drabkin,
unpublished data.
25223
Human Initiator MethioninetRNA A.
I
40
I n V
-I
2
I-
20
H
0 0
FIG. 3. RPC-5 analysis of [36Sl Met-tRNA obtained from CV-1 cells infected with recombinant SV40 virus. A, wild type initiator tRNA;B , T54 mutant C, T60 mutant;D, T54T60 double mutant.
20
40
00
FractionNumbrr
Fraction Number
C.
D.
40
0
w
20
-
T54T60
0 A
s
-I
I-
-
I n
I n
2
40
WT
T60
20
+
w
WT 0
0
20
40
FractionNumbrr Aminoacylation of Total tRNAs with Methionine-Analytical aminoacylation of total tRNA using crude E. coli synthetases was performed as described by Stanley (1974) in a 100-p1 mixture containing 50 mM sodium cacodylate, p H 7.4,15 mM MgC12, 10 mM ATP, 1 mM CTP, 3-5 pg of tRNA, 10mM [35S]methionine(specific activity between 7 and 12 Ci/mmol), and 5 p1 of the enzyme preparation (113 pg) a t 37 "C. Under the buffer conditions used here, the elongator methionine tRNA is not aminoacylated to any detectable level (Stanley, 1974; Drabkin and RajBhandary, 1985b). Analytical aminoacylations using rabbit liver aminoacyl-tRNA synthetaseswere performed in a similar manner except that the MgCl, concentration was 10 mM and 120 pg of enzyme were used (Stanley, 1974). In both cases, 5-pl samples were withdrawn for determination of trichloroacetic acidinsoluble material according to the method of Bollum (1968). Each preparation of tRNA was also assayed for leucine acceptance using the rabbit liver enzyme and [3H]leucine. The leucine acceptance of the various tRNA preparations did not differ significantly. Preparative Aminoacylation of the Gel-purified tRNAs with P'S] Methionine-Thiswas performedessentiallyas describedfor the analytical reactions, except that 6.8 mM (280 Ci/mmol) [35S]methionine, 1-2 pg of tRNA, and 0.8 pg of purified E. coli methionyl-tRNA synthetase were used. Preparative labeling of the elongator methionine tRNA was done using the crude rabbitliver enzyme. After aminoacylation, the reactions were terminated by addition
60
E
a! L,,
0
20
40
00
FractionNumber
of 0.3 volume of 1 M sodium acetate, pH 4.5, and 1 volume of phenol saturated with 0.3 M sodium acetate. After mixing, the phases were separated by centrifugation. The organic phase and interphase were re-extracted once with 0.1 M sodium acetate, pH 4.5, as above, and 2.5 volumes of ethanol were added to the combined aqueous phases. The RNA was collected by centrifugation, dissolved in 10mM sodium acetate, pH4.5, and free [35S]methionine and excess nucleotides were removed by dialysis against 0.5 M NaC1, 50 mM sodium acetate, pH 4.5, and then against10 mM sodium acetate, pH4.5, or in some cases by multiple ethanol precipitations. Synthetic mRNAs Used for in Vitro Translations-pSpa was obtained from L. Gerhke (Jobling and Gehrke, 1987). This is an Sp6 construct carrying the rabbit a-globin cDNA gene and an AMV4 leader sequence. pSpA was constructed from pSpaby removal of the NcoI-AccI fragment,and replacing itwiththe sequence CATGAAGGCCCTGT (see Fig. 7). This removes 246 base pairs of DNA coding for amino acids 1-82 of a-globin and replaces them with the sequence Met-Lys-Ala-Leu. The internal methionine is removed in this process. pSpNS, carrying the VSV NS gene, was a gift ofA. Banerjee (Gill etal., 1986). I n vitro transcriptions were carried out in the presence of cap analog using Sp6 RNA polymerase essentially as described by Drabkin (1988). Cell-free Translations-Activity of the mutant tRNAs in initiation was measured in nuclease-treated reticulocyte lysates (Promega), or
Human InitiatorMethionine tRNA
25224
@
I
CUACCAon AUCGp
2 ~'AAUCCAUCCUCUG~
G I : C 7 2 / A 2 9 T 31 .. : T39T41 / T54IT60
tRNA i
fa FIG.4. T-1RNase fingerprint analysis ofin vivo S2P-labeledtRNAs. Left, wild type initiator tRNA;right, Gl:C72/A29T31:T39T41/ T54T60 mutanttRNA. wheat germ lysates (Sachs et aL, 1989). Reticulocyte lysate incubations (25pl) contained 16 pl of reticulocyte lysate, 20 p~ each of the unlabeled amino acids, except methionine, which was 1 mM, 80 mM potassium acetate, 0.5 mM magnesium acetate, 2 mM dithiothreitol, 10 mM creatine phosphate, 1 pg/ml creatine kinase, 20 rnM hemin, and 70,000-130,000 cpm of [3sS]Met-tRNAs (4.01 X lo5cpm/pmol). t the Incubation was at 30 "C. Aliquots (3 p1) were removed atimes indicated and added directly to 0.2 ml of 0.2 N KOH, 2% casamino When all time acids on ice, to which were added 10 pl of 30% H202. points were collected, the samples were incubated at 37 "C for 10 min, after which 0.25ml of 10%trichloroacetic acid was added. The samples were mixed, left on ice for 15 min, filtered through glass fiber filters (Schleicher & Schuell no. 30), dried, and counted in Liquiscint scintillation fluid (National Diagnostics).
-
-
0
m
p'6A *PC
*pG
$
*pu
Prlr
a
e
'-'
5. anticodon fragment ( B )initiator tRNAs.
Of
digestion Of the T-lRNase-derived wild type ( A ) and A29T31:T39T41
TABLE I
RESULTS
Mutants of Initiator tRNA-The mutants generatedfor this study (Fig. 1) can be divided according to the region of the tRNA mutated. First, theAl:U72 base pair at the endof the acceptor stem is changed to Gl:C72, which is found in most other eukaryotic elongator tRNAs including the mammalian elongator methionine tRNA (Sprinzl et dl., 1991). Second, two of the three consecutive G:C base pairs found in the anticodon stem are changed tosequences found in the mammalian elongator methionine tRNA. The 931:939 base pair is unique to the eukaryotic elongator methionine tRNA, although several elongator tRNAs have A31:'k39 (Sprinzl etal., 1991). Third, the A54 and A60 residues in loop IV found in all eukaryotic initiators was changed to T54 and T60. Although C60 is mostoftenfoundinelongatormethionine tRNAs, we changed A60 to T60 to study the effects of potentially extending the length of the T9CG stem when A54 is present onexpression of the mutant tRNAs. Expression of Mutant tRNAs in CV-1 Cells-CV-1 cells infected for 48 h with the various recombinant virus stocks were labeled for 5 h with [32P]orthophosphate.Nucleic acids were isolated, and the tRNAs were analyzed on 15% polyacrylamide (19:l acry1amide:bisacrylamide) 7 M urea gels run at 500 V as described previously (Drabkin and RajBhandary, 1985b). Fig. 2 shows a n autoradiogram of a typical analysis.
*
Aminoacylntion as a measure of overexpression of mutant tRNAs Overexpression is measured as either a ratio of the amount of initiator methionine tRNA in the indicated culture to the initiator methionine tRNA in a mock-infected culture, or the ratio of mutant initiator methionine tRNA to the endogenous initiator methionine tRNA separated by RPC-5 chromatography (see Fig. 3 for an exarnde). Mutant
Relative overexpression
12.5 (2.3)" Wild type 14.7 (0.4) Gl:C72 10.4 (2.3) T54T60 7.3 (1.6) A29T31:T39T41 7.5 (0.3) Gl:C72/T54T60 6.7 (2.6) A29T31:T39T41/T54T60 7.9 Gl:C72/A29T31:T39T41 4.5 (1.1) Gl:C72/A29T31:T39T41/T54T60 a The number in parentheses indicates the standarddeviation when several independent preparations are analyzed.
Each sample from cells infected with virus carrying a tRNA gene (lanes 2-8) shows a prominently labeled band not present in RNAfrommock-infected cells (lane 1). Allof the mutant tRNAgenes appear tobe expressedto a similar extent during the 5-hlabeling period. Fig. 2 also shows that theelectrophoretic mobility of some
25225
Human Initiator Methionine tRNA
WT
I-
/ O
5
10
15
TIME (minuten)
20
25
5
A29TSl:TSBT41 A29TSl:TSBT41
10
15
20
25
TIME (minuten)
FIG.6. Activity of wild type and mutant [35S]Met-tRNAsin initiation in a reticulocyte cell-free system programmed with the SpNS mRNA.
Characterization of the Mutant tRNAs-The sequence of each of the mutant tRNAs was verifiedby fingerprint analysis of the T1-RNasedigestion products by two-dimensional homochromatography. Fig. 4 compares the fingerprints of the wild '., Nl IW o* IU 7 type initiator tRNA and the Gl:C72/A29T31:T39T41/ S C ~ ~ ~ B. TCCCGGGACAGAJ' T54T60 mutant tRNA carrying eightnucleotide changes. All be FIG. 7 . Schematic diagram of the constructionof SPA. A , the of the expected nucleotide changes can seen. First, the oligonucleotide pAGp, present in the fingerprint original Spa cDNA; B , the synthetic duplex which replaced the NcoIAccI fragment outlined in A. of the wild type tRNA, is absent in the fingerprint of the Gl:C72/A29T31:T39T41/T54T60 mutant, and in its place, of the mutant tRNAs is altered, as seen previously for the the oligonucleotide pGp is found. Second, the oligonucleotide T54 mutant (Drabkin and RajBhandary, 198513). The T54T60 CUACCAOH, seen in the wild type is replaced in the mutant mutant (lane 3 ) moves somewhat slower than the wild type with CCACCAOH. The presenceof these oligonucleotides dema G1:C72 base pair tRNA (lane2). The G1:C72 mutant (lane4 ) moves essentially onstrates that the mutant tRNA contains the same as the wild type tRNA, whereas the A29T31:T39T41 and is correctly processed at both 5' and 3' ends. Third, the mutant (lane 5 ) moves significantly faster than thewild type oligonucleotidesCUG and CCCAUt'AACCCAUG, derived tRNA. Coupling additional mutations appears t o restore the from the anticodon stem and loop regions of the wild type of the mobility to that of the wild type initiator tRNA. The human initiatortRNA,arenotpresentinthefingerprint mutant tRNA. Instead, the oligonucleotide CUAG, consistent elongator tRNA has a mobility significantly slower than the wild type initiator tRNA under these conditions. Therefore, with the change of G29 to A29, is found. In addition, the oligonucleotide qCCCAUt'AA9CUAUG is found inthe these mobilitydifferences can be exploited to separate, in of CCCAUt' many cases, the mutant initiator tRNA from the endogenous fingerprint of themutanttRNAinstead of the wild type initiator wild type initiator tRNA without contamination from the AACCCAUG found in the fingerprint tRNA. Fourth, the oligonucleotides AUCG and elongator methionine tRNA. m'AAACCAUCCUCUG, derived from loop IV loop and the Since the Gl:C72 mutant tRNA could not be separated adjacentstem sequenceleading totheacceptorstem,are from wild type initiator tRNA by gel electrophoresis,the extent of contamination of this mutant tRNA withwild type changed toT W G and m'AAUCCAUCCUCUG in thefingerinitiator methionine tRNA was estimated from the extent of print of the mutant tRNA. Similar analysis was performed overexpression of the mutant tRNA as measured by amino- for every mutant and all of the expected base changes were observed. acylation. The extent of contaminaton was found to be at In addition to theabove analyses, the extent of nucleotide of gel-purified T54T60, G1:C72/ most 10%. Thepurity T54T60, Gl:C72/A29T31:T39T41, and Gl:C72/A29T31: modification was investigated by cbromatogrnphy of total T39T41/T54T60 mutant tRNAs were estimated by amino- nuclease P1 or T2-RNase digests of each mutant tRNA on thin layer plates (Nishimura, 1972). The digests were peracylation followed by separation of wild type and mutant formed on either the whole tRNA, or in some cases, on an Met-tRNAs on RPC-5 columns. For the T54T60 mutant, this isolated oligonucleotide produced by T1-RNase digestion. Fig. was found to be88% (Fig. 3 0 ) .
25226
Human InitiatorMethionine tRNA
30
I
A29T31:T59T41
n W
K K
20
ul
z
a+ 3i
a 0
L1
i / /
10
0 40
1
I
10 30
20
I
J
0
TIME (minutes)
/ A20T31:T39T41 /T54T60
I
I
30 10
20 TIME (minutes)
I
I
40
FIG. 8. Activity of wild type and mutant i%lMet-tRNAs in initiation in a wheat germ cell-free system programmed with the SPA mRNA. "
"
5shows theresults of a comparative nuclease P1 digest the mutant tRNAs to accept methionine was studied using performed on the isolated T1-RNase fragment derived from both E. coli and rabbit liver aminoacyl-tRNA synthetases. the anticodon region of the wild type and A29T31:T39T41 Table I summarizes the results of aminoacylation reactions mutant initiator tRNAs. for all of the mutants. using theE. coli enzyme and total tRNA Pseudouridine is found in the anticodon stemonly when it The following conclusions can be drawn. First, all mutant tRNAs are aminoacylated with methionine has the sequence found in the elongator methionine tRNA (Panels A versus B ) . Furthermore,, the intensity of the pseu- by the E. coli enzymes. This is interesting, since the mamdouridine 5"phosphate spot in the nuclease P1 digests is malian elongator methionine tRNA is not aminoacylated by stronger than the pt6A spot, as expected for the presence of the E . coli methionyl-tRNA synthetase under the conditions 1974; Drabkinand two \k residues compared t o one t6A. Analysis of mutant used intheseexperiments(Stanley, tRNAs carrying the T54 mutationshows that, in addition to RajBhandary, 1985b). All of these mutant tRNAs are also conversion of U54 to T54,U55 is converted to q55, consistent aminoacylated by the rabbit liver enzyme (data not shown). It should be noted that the aminoacylation assayswere perwithourearlierobservations(DrabkinandRajBhandary, formed using enzyme excessand total tRNA for measurement 1985b) (data not shown). We oftenobserve partial modification of A58 to m'A58, and of tRNA levels. Therefore, we cannot comment on the effiA37 to t6A37, as evidenced by the occurrence of additional ciency of aminoacylation of the different tRNAs. In addition, oligonucleotides in T1-RNase fingerprints (Fig. 4). Under- it is not known if any of the mutations introduced lead to modification of the mutant tRNAsmay be due to any one or tRNAs that can be misacylated by other aminoacyl-tRNA a combination of the following: 1)the inability of the pool of synthetases. Second, the extent of overproduction of mutant tRNAs co-factors and enzymes to adequately meet the demand of the ranges from 5- to 15-fold over endogenous levels of initiator levels of tRNAbiosynthesis achieved during infection;2) effect of mutations on recognition of the mutant tRNAs by tRNA duringa 48-h infection. Thewild type and the Gl:C72 the modifying enzymes; 3) effects of tRNA structure/sequence and the T54T60 mutant tRNAs consistently give at least a give alterations onnuclear transport of the tRNA. Thismay be of 10-fold overexpression,whereas the other mutant tRNAs between 5- and 7-fold overexpression. The reasons for these importance formodificationswhichoccur within the cytoplasm rather than the nucleus. Thesemodifications seem differences are not known, and could be due to effects of the mostly to be those occurring in the anticodon loop; and 4) mutations on transcription, processing, transport of tRNA effects of SV40 infection on biosynthesis of modifying en- from thenucleus, or stabilityof the transcripts, although none zymes and/or their stability. Forexample, a unique species of of the mutations madewould be predicted toadversely influlysine tRNA is found in SV40 transformed mouse cells. This ence any of these processes. tRNAs carrying single mutations in loop IV appear to be tRNA is a hypo-modified tRNA?, in which a dihydrouridine in loop I, and t6A in the anticodon loop are missing (Raba et present at a lower level compared to other mutant tRNAs. This is shown in more detail in Fig. 3. The T54 mutant,with al. 1979). Aminoacylation Assays on Mutant tRNAs and Measurement a potential U54:A60 base pair, is expressed about one-third of Extent of Their Expression in CV-1 Cells-The ability of as well as the wild type initiator tRNA (Drabkin and Raj-
Human Initiator Methionine tRNA
25227
Bhandary, 1985b). The T60 mutant, with a potential A54:U60 at its 5' end the leader sequence of alfalfa mosaic virus 4, signal in base pair, is expressed even less well. When both mutations which constitutes a strongtranslationinitiation are combined to T54T60, however, the expression level is wheat germ extracts. The alfalfa mosaic virus 4 leader sequence is followed by a sequence coding for amino acids82about equal to that of wild type tRNA (TableI ) . 142 of rabbit a-globin. The SpA was constructed by replacing It is interesting that the T54T60 mutant tRNA is expressed as well as the wild type initiator tRNA. Aspecial feature a 242-base pair NcoI-AccI fragment which encodes the first observed in the three-dimensional structureof yeast initiator 82 amino acids of the rabbit a-globin, including the only tRNA is an intricate network of hydrogen bonds involving internal methionine, with a short synthetic NcoI-AccI fragA20 in loop I and m'A58 and A60 in loop IV (Basavappa and ment coding for four amino acids, the second of which is Sigler,1991). Two of thesenucleotides A20 and A60 are lysine instead of valine. This results in a 64-amino acid long unique t o eukaryotic initiator methionine tRNAs. These ad- protein, which contains a single methionine residue at its N followed by a lysine, ditional hydrogen bonds augment the other tertiary interac- terminus. The N-terminal methionine is tions within loop IV and between loop I and loop IV seen in and is, therefore, resistant to N-terminal methionine aminoelongator tRNAs. Thehigh level of expression of the T54T60 peptidase (Tsunamaet al., 1985). Transfer of methionine into mutant initiator tRNA suggests that these additional inter- protein directedby this message therefore measures initiation. In general, theeffect of mutations on initiation activity is actions between A20, m'A58, and A60 seen in the initiator tRNA structure are not essential for the overall folding of the less severe in the wheatgerm system than in thehomologous mammalian reticulocyte system. Mutationof the Al:U72 base tRNA and its stability in mammaliancells. Overall, the mutant tRNAs arewell expressed considering pair toa Gl:C72 base pairproduces a tRNA withsignificantly the fact that the endogenous levels of initiator tRNA repre- lower activity in initiation (Fig. 8, left panel). Mutations in the G:C base pairs in the anticodon stem have only a modest sent the combined output of at least 10-13 initiator tRNA genes in mammalian cells (Santos and Zasloff, 1981). The effect on initiation. This result is in contrast to the effect of reticulocyte system where the levels of overexpression do not differ more than 3-fold. Analy- thesamemutationsinthe mutant tRNAwas essentially inactive in initiation. Mutations sis of Hirt (1967) extracts of the nuclear pellets obtained during workup of the large scale preparations shows similar of A54 and A60 to T54 and T60also produces a tRNA with reductioninactivity in initiation.Mutant levels of infection with viruses carrying the different mutant onlyamodest tRNAs inwhich the various mutations are coupled are signifinitiator tRNA genes. The Activityof Mutant tRNAs in Initiation-We have used icantly less active in initiation than the individual mutants two different protein synthesis systemsto study the effects of (Fig. 8, right panel),suggesting a cumulative effect of each of themutationsoninitiation.The double mutantcarrying the above mutations on their overall activity in initiation. The first system used a rabbit reticulocyte lysate programmed changes in the Al:U72 base pair and in the G:C pairs in the (Fig. 8, with Sp6 transcripts coding for the VSV NS protein (Gill et anticodonstem is essentiallyinactiveininitiation al., 1986). The assay measures the transfer of [3sS]methionine right panel). from the various [3sS]methionyl-tRNAs into protein.The NDISCUSSION terminal methionine of VSV NS protein is stable, therefore, transfer of [3sS]methionineintoproteindirected by this A clear result of these studies is that changing theAl:U72 mRNA provides adirect measureof the activityof the various base pair in the amino acid acceptorstem of the human mutant tRNAs in initiation. Although the VSV NS protein initiator tRNA significantly decreases its activity in initiation has internal methionines, participationof any of the mutant in reticulocyte and wheatgerm cell-free systems. Thus, asfor initiator tRNAs in elongation unlikely, is since similar assays the E. coli initiator tRNA, nucleotides 1 and 72 at the endof programmed with Spa mRNA,coding for rabbit a-globin, did theacceptorstemareimportantininitiation(Seongand not show any incorporation of methionine. RajBhandary, 1987b). With the E. coli initiator tRNA, the Mutations of the Al:U72 base pair to a G1:C72 base pair ClxA72 mismatchis important for three of its four distinctive has a strong effect on activity in initiation(Fig. 6, left panel). properties. These include formylation of the tRNA,exclusion The slight incorporation seen is most likely due to contami- of the tRNA from the ribosomal A site, and preventing the nation of the mutant tRNA by some wild type initiator tRNA. tRNA from being a substrate for peptidyl-tRNA hydrolase Mutations in the conserved G:C base pairs in the anticodon (Varshney et al., 1991; Lee et al., 1992). With the human stem alsoproducea tRNAthatisessentiallyinactivein initiator tRNA, it is possible that the Al:U72 base pair is initiation (Fig. 6, right panel). Mutations of A54 and A60 to important eitherfor its specific binding to the initiation factor T54 and T60lowers activity in initiation somewhat, althougheIF-2:GTP, and/or for its binding to the P site on the ribothe effect is much less pronounced than that of mutations a t some. The importance of the Al:U72 base pair in function in the other two sites (Fig. 6, left panel). Thus, mutations in uiuo of the yeast initiator tRNA hasalso been demonstrated each of the conserved sequences in eukaryotic initiator tRNAsrecently by Bystrom and co-workers (von Pawel-Rammingen affect their overall activity in initiation.As expected from the et al. 1992). It is possible thatwhat is importantisnot above results, the mutant with changes in all three features specifically the presence of an Al:U72 base pair, but a weak is essentially inactive in initiation(Fig. 6, left panel). base pair between nucleotides 1 and 72. While the Al:U72 The second protein synthesis system used wheat germ cell- base pair is highly conserved among all eukaryotic initiator free extracts. Although this system is heterologous with re- tRNAs, the tRNA from Schizosaccharomycespombe is an spect to the mutant tRNAs being tested, the wheat germ exception in that it hasa $1:A72 base pair (Keith et al., 1993; initiator tRNA also has the same conserved features present Mao et al., 1980). Also mutations of the Al:U72 base pair to in all eukaryotic initiator tRNAs, including the C33 found in Ul:A72 in yeast initiator tRNA produce a tRNA that is still the human initiator tRNA. Furthermore, the wheat germ and active in initiation (von Pawel-Rammingen et al., 1992). human initiator tRNAs have comparable activities in this Changesintheanticodonstemalso greatlyreduce the system (data not shown). activity of the mutant tRNA in initiation inreticulocyte The mRNAused for thispurpose was SPA (Fig. 7), a lysates. This result is also similar to that obtainedfor the E. deletion derivative of Spa (Jobling and Gehrke,1987). It has coli initiator tRNA. In E. coli, mutations in these G:C base
25228
Methionine InitiatorHuman
tRNA
pairs affect specifically the binding of the mutant tRNAs to tRNAs in yeast (Francis and RajBhandary,1990). The ready the ribosomal "P"-site (Seong and RajBhandary,1987a). separation of the human initiator tRNA from endogenous the The less pronounced decrease in activity of the same mu- yeastinitiatortRNAonRPC-5columns(Drabkinand tant initiator tRNA in the wheat germ system could be due RajBhandary, 1985a; Francis and RajBhandary,1990) should to the use of a heterologous protein synthesis system. Alter- facilitate large scale purification of the mutant tRNAs. Studprogress. natively, the presenceof three consecutive G:C base pairs in ies along these lines are in the anticodon stem of the initiator tRNA may not be as Acknowledgment-We thank P. Sharp for making tissue culture critical for fungal and plant protein synthesis systemsas for facilities available. a mutant yeast strain mammalian systems. For example, using REFERENCES carrying only one functional yeast initiator tRNAgene (Bys- Basavappa, R., and Sigler,P. B. (1991) EMBO J. 1 0 , 3105-3111 trom and Fink, 1989),von Pawel-Rammingen et al. (1992) Bollum, F. J. (1968) Methods Enzymol. 1 2 , 169-173 Bystrom, A. A,, and Fink, G. R. (1989) Mol. Cen. Geuet. 2 1 6 , 276-286 havereplaced the residual tRNA gene with mutant yeast Drabkin, H. J. (1988) Nucleic Acids Res. 16,11591-11606 Drabkin, H. J., and RajBhandary,U. L. (1985a) J. Bid. Chem. 260,5596-5602 initiator tRNA genes and have shown that the Drabkin, H. J., and RajBhandary, U. L. (1985b)J. Biol. Chem. 260,5588-5595 A29T31:T39T41 mutantyeastinitiatortRNAgene could Drabkin, H. J., and RajBhandary, U. L. ( 1 9 8 5 ~J. ) Bwl. Chem. 260,5580-5587 support growth of the yeast strain. It should be noted, how- Francis, M., and RajBhandary, U. L. (1990) Mol. Cell. Biol. 10,4486-4494 D. S., Chattopadhyay, D., and Banerjee, A. K. (1986) Proc. Natl. Acad. in uiuo system the activity of the Gill, ever, that in the yeast Sci. U. S. A . 83,8873-8877 Hirt, B. (1967) J. Mol. Biol. 2 6 , 365-369 mutant initiator tRNAs is being studied in the absence of any S. A,, and Gehrke, L. (1987) Nature 325,622-625 wild type tRNA as a competitor. In contrast, both cell-free Jobling, Keith, G., Heitzler, J., El Adlovni, C., Glasser, A,-L., Fix, C., Desgres, J., and Dirheimer, G. (1993) Nucleic Acids Res. 2 1 , 2949 systems used in the present study support protein synthesis Kozak, M. (1983) Microbiol. Reu. 4 7 , 1-45 intheabsence of anyaddedtRNA;themutantinitiator Kozak, M. (1992) Annu. Reu. Cell Biol. 8 , 197-225 Kramer, W., Drutsa, V., Jansen, H.-W., Kramer,B., Pflugfelder, M., and Fritz, tRNAs are, therefore, competing with endogenous initiator H.-J. (1984) Nuclerc Actds Res. 12,9441-9456 tRNA present in the extracts. Lee, C. P., Dyson, M. R., Mandal, N., Varshney, U., Bahramian, B., and Of the changes introduced in the three regions of the human RajBhandary, U. L. (1992) Proc. Natl. Acad. Sci. U. S. A . 89,9262-9266 Leontis, N., DaLio, A,, Strobel, M., and Engelke, D. (1988) Nucleic Acids Res. initiator tRNA, the one with the least detrimental effect in 16,2537-2552 K. M., Jones, S. S., Hackett, N. R., and Khorana, H. G. (1984) Proc. Natl. in uitro initiation is the T54T60 mutation in loop IV. This is Lo,Acad. Sci. U. S. A . 8 1 , 2285-2289 not to say that this mutation has noeffect in initiation (Fig. Mao, Jen-I., Schmidt,O., and Soll, D. (1980) Cell 2 1 , 509-516 Merrick, C. (1992) Microbiol. Reu. 5 6 , 291-315 6, left,and Fig. 8, right). However, the significant activity of Moldave, W. K. (1985) Annu. Reo. Biochem. 5 4 , 1109-1149 this mutant tRNA in initiation in uitro contrasts with the Nishimura, S. (1972) Prog. Nucleic Acid Res. Mol. Biol. 1 2 , 49-85 R. L., Hancher, C. W., Weiss, J. F., Holladay, D. W., and Kelmers, A. finding of Bystrom and co-workers (von Pawel-Rammingen Pearson, D. (1973) ,Biochim. Biophys. Acta 294,236-249 et al., 1992) that mutant yeast initiator tRNAs carrying the Raba, M:, Lmburg, K., Burghagen, M., Katze, J. R., Simsek, M., Heckman, J. E., RaJBhandary, U. L., and Gross, H. J. (1979) Eur. J. Biochem. 9 7 , 305T54 mutation, including the T54C60 double mutation, are 318 inactive in yeast. It is possible, although unlikely, that the RaJBhandary, U. L., and Ghosh, H. P. (1969) J. Biol. Chem. 244,1104-1113 Sachs, M. S., Bertrand, H., Metzenberg, R. L., and RajBhandary, U. L.(1989) difference between these results is due to change of A60 to Mol. Cell. Biol. 9, 566-577 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sei.U. S. A . C60 in the T60 in the human initiator tRNA mutant and 74,5463-5467 yeast initiator tRNA mutant. An alternative possibility is Santos, T.,and Zasloff, M.(1981) Cell 23,699-709 that, in yeast, theT54C60 mutant tRNA issomehow seques- Seong, B. L., and RajBhandary, U. L. (1987a) Proc. Natl. Acad. Sci. U. S. A . 84,334-338 tered away from the initiation factor eIF-2. It isknown that Seong, B. L., and RajBhandary, U. L. (1987b) Proc. Natl. Acad. Sci. U. S. A . 84,8859-8863 the T54T60 andT54C60 mutations do not affect levels of the Simsek, M., and RajBhandary, U. L. (1972) Biochem. Biophys. Res. Commun. mutant tRNAs in uiuo in CV-1 cells (Table I) and in yeast, AQ """ wr-61K Simsek, M., Ziegenmeyer, J., Heckman, J., and RajBhandary,U. L. (1973) Proc. respectively. Natl. Acad. Sci. U. S. A. 7 0 , 1041-1045 Finally, identification of the precise role of each of the Sprinzl, M., Dank, N., and Nock, S. (1991) Nucleic Acids Res. 19, 2127-2171 Stanlev. W. M. (1974) Methods Enrvmol. 29. 530-547 special features of the initiator tRNA on initiation will require Tsunsiwa, S., Stewart, J. W., and"Sherman, F. (1985) J. Biol.Chem. 2 6 0 , detailed biochemical analysis of the mutant tRNAs, including 5382-5391 U., Lee, C. P., and RajBhandary, U. L. (1991) J. Biol. Chem. 2 6 6 , studies on their binding to initiation factor eIF-2 and to the Varshney, 24712-24718 P site on the ribosome. This in turn requires the availability von Pawel-Rammineen. U.. Astrom.. S... and Bvstrom. A. S. (1992) Mol. Cell. Biol. 1 2 , 1432-14x2 of mutant tRNAs inlarger quantities than that generatedby Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., and Itakura, K. (1979) Nucleic Acids Res. 6,3543-3557 expression of mutant tRNA genes in mammalian cells and W. I., Gitschier, J., Lasky, L. A,, and Lawn, R. M. (1985) Proc. Natl. free of wild type initiator tRNA. Toward this end, we have Wood, Acad. Set. U. S. A. 8 2 , 1585-1588 developed a system for expression of mutant human initiator Zoller, M. J., and Smith,M. (1982) Nucleic Acids Res. 10,6487-6500
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